For the gravimetric calibration of pipettes high resolution precision balances, analytical balances, semi micro balances, micro balances or ultra micro balances (hereinafter referred to as balances) are used. When conducting the gravimetric calibration of the nominal volume of pipettes with the aid of a balance, a volume of liquid that is to be determined is dispensed from the pipette tip into a weighing vessel; and the weighing value is used to determine the volume of the quantity of liquid that was dispensed. In this respect it is known to consider additional parameters, such as the air temperature, the liquid density, the air humidity and the air pressure, since these parameters affect the weighing result. For example, the air temperature and the air humidity have an effect on the evaporation rate of the sample liquid.
According to standard operating procedures, volumes of up to 1 μl are calibrated. Especially with very small volumes the effect of the liquid evaporation (and, as a result, the error) on the weighing result and on the accuracy of the calibration of the pipette should not be considered to be negligible.
In order to minimize the evaporation of the sample liquid during the weighing operation, so-called evaporation traps, which are disposed in the weighing chamber, are used in the application of a pipette calibration process. These evaporation traps are filled with water; and the evaporation of this water causes the air volume in the weighing chamber to be highly saturated with water. As a result, it is possible to achieve a relative air humidity of up to 90%.
Nevertheless, even with the use of evaporation traps, it cannot be completely prevented that a portion of the sample liquid will evaporate during the calibration process. This is due to the fact that the pipetting operation itself leads to air movements and to an air exchange between the weighing chamber and the surrounding area, so that the saturation of the air volume fluctuates.
In addition, when using a balance, a valid measurement value cannot be obtained immediately after the pipette to be calibrated has dispensed a specified volume of liquid; instead, it is necessary to wait a certain amount of time. With respect to falsification due to the evaporation this time period should be less than 60 seconds. Experience shows that, depending on the resolution, the handling and the type of balance, the amount of time is in a magnitude of 5 to 20 seconds. It cannot be prevented that during this process time a portion of the liquid to be measured has already evaporated, and, as a result, the measurement result is falsified. This effect disproportionately affects small volumes.
Therefore, it is known in the prior art that the weighing value is corrected using an assumed rate of evaporation. Such evaporation rates have been determined experimentally for specific vessel geometries and values of the relative humidity inside the weighing chamber and range, for example, from 0.05 μg/s, when pipetting into a narrow neck flask using an evaporation trap that guarantees a relative humidity of 90% in a closed weighing chamber, to, for example, 4.6 μg/s, when pipetting into a beaker, again using an evaporation trap, which however, generates a relative humidity of less than 90% in an open weighing chamber. These values apply to distilled or deionized water of quality 3 in accordance with ISO 3696 as the pipetting liquid. For the calibration of pipettes a fixed value for the evaporation is generally assumed.
It is easy to see that the effect of the evaporation on the measurement error cannot be considered to be negligible. At an assumed evaporation rate of 0.26 μg/s, the result is an evaporation volume of 3.12 μl during a process period of 12 seconds for handling and settling the balance. The standard measurement uncertainty, according to EN-ISO 8655-6, in a measurement range of 1 μl to 10 μl is 2 μl. Therefore, the assumed evaporation is greater than the measurement error. In this case the assumed rate of evaporation of 0.26 μg/s is still a rather low value; to some extent significantly higher values have been mentioned in the literature.
In order to reduce the effect of the environment on the accuracy of the measurement, the prior art discloses a number of measures for determining the mass.
For mass comparators it is known, for example, that the air buoyancy is determined by a comparison measurement of two reference objects having a mass and density that are already known beforehand.
It is known that the temperature, the air pressure and the humidity also affect the balance itself. For this reason, in order to compensate for the variances in the weighing result with changing ambient parameters, correction factors are stored in the device, for example, in the form of curves or tables. In addition, temperature and air humidity sensors are disposed in the surrounding area of the load cell, for example, in the laboratory. Then these temperature and air humidity sensors are used to automatically correct the balance itself, as a function of the changing ambient conditions.
The European patent EP 1 975 577 A1 discloses a balance for gravimetric calibration of pipettes, which has a draft shield and a built-in temperature sensor, air pressure sensor and air humidity sensor.
The German patent DE 37 14 540 C2 describes a method for automatically calibrating a high resolution electronic balance, wherein such environmental factors as the temperature change and the humidity change, both of which are detected from the outside, are used to calibrate the balance itself. The corresponding calibration factor is determined by a computer and corrects the weighing result.
The German patent DE 299 12 867 U1 discloses an analytical balance with a measuring sensor for ambient parameters. In this case the analytical balance has a display that is provided on the rear wall of the weighing chamber. The display shows the temperature in the weighing chamber and the air humidity in the weighing chamber as well as, in general, the air pressure that is usually present. In this case it is assumed that, when the air is wet, the surface of the sample to be weighed will be covered with moisture, which is a function of the variances in the air humidity. Therefore, the operator is informed by the display that, for example, with changing air humidity the sample to be weighed should remain in the weighing chamber longer, in order to obtain a stable end value of the surface moisture. If there are extreme fluctuations in the air pressure, the operator can perform a so-called buoyancy correction by feeding the displayed data to a processor in the balance via an input unit. With respect to the temperature, this temperature is used to determine the deviation from the reference temperature and to consider corresponding correction factors.
Finally there are also climatized measuring chambers, in which there are balances. In this case the climate data, which are determined by the sensors and which relate to the measuring chamber, are entered into specific software, which determines then the corresponding correction parameters, which are fed manually or automatically into the balance.
All of these measures in themselves do not lend themselves to increasing the measuring accuracy when calibrating pipettes, because in the best case scenario the sensors are placed in the immediate vicinity of the balance, but not on or in the balance itself.